Simultaneous determination of hydrochlorothiazide and losartan potassium in tablets by high-performance low-pressure chromatography using a multi-syringe burette coupled to a monolithic column

This contribution describes use of a separation method based on on-line coupling of a multisyringe flow system with a chromatographic monolithic column for simultaneous determination of hydrochlorothiazide and losartan potassium in tablets. The system comprised a multisyringe module, three low-pressure solenoid valves, a monolithic C₁₈ column (25 mm × 4.6 mm i.d.), and a diode-array detector. The mobile phase was 10 mmol L⁻¹ potassium dihydrogen phosphate (pH 3.1)-acetonitrile-methanol (65:33:2 v/v/v) at a flow rate 0.8 mL min⁻¹. UV detection was carried out at 226 nm. The multi-syringe chromatographic (MSC) method with UV spectrophotometric detection was optimized and validated. Results from validation were very good. The analysis time was about 400 s. The method was found to be applicable to routine analysis of both compounds in tablets. The coupling of the monolithic columns with a multi-syringe flow-injection analysis manifold provides an excellent and inexpensive tool to solve the separation problems without use of HPLC instrumentation.     

Chemometrics-assisted simultaneous determination of atenolol and chlorthalidone in synthetic binary mixtures and pharmaceutical dosage forms

Resolution of binary mixtures of atenolol (ATE) and chlorthalidone (CTD) with minimum sample pre-treatment and without analyte separation has been successfully achieved, using a new and rapid method based on partial least squares (PLS1) analysis of UV spectral data. The simultaneous determination of both analytes was possible by PLS1 processing of sample absorbances between 255 and 300 nm for ATE and evaluation of absorbances in the 253–268 nm region for CTD. The mean recoveries for synthetic samples were 100.3±1.0% and 100.7±0.7% for ATE and CTD, respectively. Application of the proposed method to two commercial tablet preparations in the content uniformity test showed them to contain 103.5±0.8% and 104.9±1.8% ATE respectively, as well as 103.4±1.2% and 104.5±2.2% CTD. Use of this method also allowed the elaboration of dissolution profiles of the drugs in two commercial combined formulation products, through the simultaneous determination of both drugs during the dissolution test. At the dissolution time of 45 min specified by USP XXIV, both pharmaceutical formulations complied with the test.     

Application of a chemometric method for simultaneous determination of acetaminophen and diclofenac in content-uniformity and drug-dissolution studies

A new, repeatable, and rapid method has been developed for resolution of binary mixtures of acetaminophen and diclofenac with minimum sample pretreatment and without separation of the analytes. The method, based on the PLS1 processing of absorbance data in the UV region, was successfully used for quantification of the drug content of three tablet preparations. The results obtained were in good agreement with HPLC recovery data. The method also enabled determination of drug-dissolution profiles of these commercial tablets, by simultaneous determination of both analytes during the dissolution test.     

PLS and first derivative of ratio spectra methods for determination of hydrochlorothiazide and propranolol hydrochloride in tablets

Two new analytical methods have been developed as convenient and useful alternatives for simultaneous determination of hydrochlorothiazide (HCT) and propranolol hydrochloride (PRO) in pharmaceutical formulations. The methods are based on the first derivative of ratio spectra (DRS) and on partial least squares (PLS) analysis of the ultraviolet absorption spectra of the samples in the 250–350-nm region. The methods were calibrated between 8.7 and 16.0 mg L⁻¹ for HCT and between 14.0 and 51.5 mg L⁻¹ for PRO. An asymmetric full-factorial design and wavelength selection (277–294 nm for HCT and 297–319 for PRO) were used for the PLS method and signal intensities at 276 and 322 nm were used in the DRS method for HCT and PRO, respectively. Performance characteristics of the analytical methods were evaluated by use of validation samples and both methods showed to be accurate and precise, furnishing near quantitative analyte recoveries (100.4 and 99.3% for HCT and PRO by use of PLS) and relative standard deviations below 2%. For PLS the lower limits of quantification were 0.37 and 0.66 mg L⁻¹ for HCT and PRO, respectively, whereas for DRS they were 1.15 and 3.05 mg L⁻¹ for HCT and PRO, respectively. The methods were used for quantification of HCT and PRO in synthetic mixtures and in two commercial tablet preparations containing different proportions of the analytes. The results of the drug content assay and the tablet dissolution test were in statistical agreement (p < 0.05) with those furnished by the official procedures of the USP 29. Preparation of dissolution profiles of the combined tablet formulations was also performed with the aid of the proposed methods. The methods are easy to apply, use relatively simple equipment, require minimum sample pre-treatment, enable high sample throughput, and generate less solvent waste than other procedures. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

HPLC‐DAD method for the simultaneous determination of zofenopril and hydrochlorothiazide in oral pharmaceutical formulations

An HPLC method with DAD detection was developed and validated for the simultaneous determination of zofenopril and hydrochlorothiazide in tablets. The separation was carried out through a gradient elution using an Agilent LiChrospher C18 column (250×4.0 mm id, 5 μm) and a mobile phase consisting of (A) water–TFA (99.9:0.1 v/v) and (B) acetonitrile–TFA (99.1:0.1 v/v) delivered at a flow‐rate of 1.0 mL/min. 8‐Chlorotheophylline was used as internal standard. Calibration curves were found to be linear for the two drugs over the concentration ranges of 5.0–40 and 1.0–20 μg/mL for zofenopril and hydrochlorothiazide, respectively. Linearity, precision, accuracy, specificity and robustness were determined in order to validate the proposed method, which was further applied to the analysis of commercial tablets. The proposed method is simple and rapid, and gives accurate and precise results.     

H-point standard addition method for simultaneous determination of phenylephrine hydrochloride,chlorpheniramine maleate,and paracetamol as a ternary mixture in pharmaceutical formulations

The simultaneous analysis of a ternary mixture containing paracetamol (PAR), phenylephrine hydrochloride (PHE), and chlorpheniramine maleate (CPM) was conducted without prior separation and using an advanced spectrophotometric method. The H-point standard addition and absorbance correction methods were selected to determine the compounds, which are highly overlapped spectra in pharmaceutical formulations. The method is based on the use of three different wavelengths of 296, 272, and 227 nm for the ternary mixture. The concentration of PAR was calculated directly at 296 nm because no interferences existed. Absorbance correction method was used to remove the role of PAR at 272 and 227 nm. The concentrations of the PHE and CPM compounds in the mixture were determined by using the H-point standard addition method. The results showed that simultaneous determination of PAR, PHE, and CPM could be conducted within the range of 1–33 μg/mL, 1–23 μg/mL, and 1–36 μg/mL, respectively. The relative standard deviations for the simultaneous determination of PHE, CPM, and PAR were 0.617, 2.76, and 1.71, respectively. The proposed method was implemented successfully for the simultaneous determination of PAR, PHE, and CPM in pharmaceutical formulations.     

Multivariate optimization and validation of an analytical methodology by RP-HPLC for the determination of losartan potassium in capsules

This paper describes the optimization and validation of an analytical methodology for the determination of losartan potassium in capsules by HPLC using 2^5-1 fractional factorial and Doehlert designs. This multivariate approach allows a considerable improvement in chromatographic performance using fewer experiments, without additional cost for columns or other equipment. The HPLC method utilized potassium phosphate buffer (pH 6.2; 58 mmol L⁻¹)-acetonitrile (65:35, v/v) as the mobile phase, pumped at a flow rate of 1.0 mL min⁻¹. An octylsilane column (100 mm × 4.6 mm i.d., 5 μm) maintained at 35 °C was used as the stationary phase. UV detection was performed at 254 nm. The method was validated according to the ICH guidelines, showing accuracy, precision (intra-day relative standard deviation (R.S.D.) and inter-day R.S.D values <2.0%), selectivity, robustness and linearity (r = 0.9998) over a concentration range from 30 to 70 mg L⁻¹ of losartan potassium. The limits of detection and quantification were 0.114 and 0.420 mg L⁻¹, respectively. The validated method may be used to quantify losartan potassium in capsules and to determine the stability of this drug.     

Simultaneous kinetic-spectrophotometric determination of carbidopa, levodopa and methyldopa in the presence of citrate with the aid of multivariate calibration and artificial neural networks

The kinetic methodology based on the difference of reaction rates, is based on the reaction between a common oxidizing agents such as tris(1,10-phenanthroline) and iron(III) complex (ferriin, [Fe (phen)₃]³⁺) in the presence of citrate and spectrophotometrically, monitoring the changes of absorbance at the maximum wavelength of 511 nm. Experimental conditions such as pH, reagents and citrate concentrations were optimized, and the data obtained from the experiments were processed by several chemometric approaches, such as artificial neural network (ANN) and partial least squares (PLS). A set of synthetic mixtures of carbidopa (CD), levodopa (LD) and methyldopa (MD) was evaluated and the results obtained by the applications of these chemometric approaches were discussed and compared. It was found that the back propagation artificial neural network (BP-ANN) method afforded better precision relatively than those of radial basis function artificial neural networks (RBF-ANN) and PLS. The proposed method was also applied satisfactorily to the determination of carbidopa, levodopa and methyldopa in real samples.     

Simultaneous determination of aliskiren and hydrochlorothiazide from their pharmaceutical preparations using a validated stability‐indicating MEKC method

A stability‐indicating MEKC method was developed and validated for the simultaneous determination of aliskiren (ALI) and hydrochlorothiazide (HCTZ) in pharmaceutical formulations using ranitidine as an internal standard (IS). Optimal conditions for the separation of ALI, HCTZ and its major impurity chlorothiazide (CTZ), IS and degradation products were investigated. The method employed 47 mM Tris buffer and 47 mM anionic detergent SDS solution at pH 10.2 as the background electrolyte. MEKC method was performed on a fused‐silica capillary (40 cm) at 28°C. Applied voltage was 26 kV (positive polarity) and photodiode array (PDA) detector was set at 217 nm. The method was validated in accordance with the ICH requirements. The method was linear over the concentration range of 5–100 and 60–1200 μg/mL for HCTZ and ALI, respectively (r²>0.9997). The stability‐indicating capability of the method was established by enforced degradation studies combined with peak purity assessment using the PDA detection. Precision and accuracy evaluated by RSD were lower than 2%. The method proved to be robust by a fractional factorial design evaluation. The proposed MEKC method was successfully applied for the quantitative analysis of ALI and HCTZ both individually and in a combined dosage tablet formulation to support the quality control.     

A validated stability-indicating LC method for simultaneous determination of quinapril and hydrochlorothiazide in pharmaceutical samples

A stability-indicating liquid chromatographic method was developed and validated for simultaneous determination of quinapril and hydrochlorothiazide in drug substances and dosage forms. Chromatographic separation of quinapril, hydrochlorothiazide and its degradation products was achieved on a RP-18 column, using acetonitrile and phosphate buffer (pH 4.6) as mobile phase in a gradient mode and detection at 216 nm. Stress testing was performed under hydrolytic, oxidative, thermal and photolytic conditions. The degradation products were well resolved from main peaks, proving the stability-indicating power of the method. The assay was linear for quinapril and hydrochlorothiazide concentrations of 40–200 µg mL^?1 and 25–125 µg mL^?1, respectively. The developed method was selective, accurate and precise for quinapril and hydrochlorothiazide determination. This method was used to quantify both drugs in combined commercial tablets. The results showed that the proposed method was found to be suitable for quantitative determination and the stability study of quinapril and hydrochlorothiazide in pharmaceutical samples.      

Simple flow-injection system for the simultaneous determination of nitrite and nitrate in water samples

A novel spectrophotometric reaction system was developed for the determination of nitrite as well as nitrate in water samples, and was applied to a flow-injection analysis (FIA). The spectrophotometric flow-injection system coupled with a copperised cadmium reductor column was proposed. The detection was based on the nitrosation reaction between nitrite ion and phloroglucinol (1,3,5-trihydroxybenzene), a commercially available phenolic compound. Sample injected into a carrier stream was split into two streams at the Y-shaped connector. One of the streams merged directly and reacted with the reagent stream: nitrite ion in the samples was detected. The other stream was passed through the copperised cadmium reductor column, where the reduction of nitrate to nitrite occurred, and the sample zone was then mixed with the reagent stream and passed through the detector: the sum of nitrate and nitrite was detected. The optimised conditions allow a linear calibration range of 0.03–0.30 μg NO₂⁻-N ml⁻¹ and 0.10–1.00 μg NO₃⁻-N ml⁻¹. The detection limits for nitrite and nitrate, defined as three times the standard deviation of measured blanks are 2.9 ng NO₂⁻-N ml⁻¹ and 2.3 ng NO₃⁻-N ml⁻¹, respectively. Up to 20 samples can be analyzed per hour with a relative standard deviation of less than 1.5%. The proposed method could be applied successfully to the simultaneous determination of nitrite and nitrate in water samples.     

2,3-Dimercaptosuccinic acid self-assembled gold electrode for the simultaneous determination of epinephrine and dopamine

Simultaneous determination of epinephrine(EP)and dopamine(DA)at 2,3-dimercaptosuccinic acid(DMSA)modified electrode was studied.The oxidation peaks of the mixture of EP and DA appeared at the same potential,but the cathodic peak currents were only linear to the concentration of DA,whereas the anodic peak currents were equal to the sum of individual anodic peak currents of EP and DA.Therefore,a novel electrochemical method for the simultaneous determination of EP and DA at a DMSA modified electrode(DMSA/A...     

Rapid and selective method for the spectrophotometric determination of tin using potassium ethylxanthate

Tin (II) forms a yellow complex with potassium ethylxanthate which can be extracted into chloroform. Tin is determined spectrophotometrically by measuring the absorbance at 360 nm. Beer's law is obeyed up to 120 g of Sn in the aqueous phase with a Sandell sensitivity of 0.013 g Sn/cm². Metal ions such as Ti(IV), Cr(VI), Mn(II), Cu(II), Zn(II), Al(III), U(VI), W(VI), Th(IV) and Zr(IV) do not interfere, but Mo(VI), Co(II) and Bi(III) do. Interference due to Fe(II, III), Ni(II) and V(V) is checked by suitable masking agents. Analysis of some synthetic and industrial samples has been carried out by the proposed method.     

A novel poly(taurine) modified glassy carbon electrode for the simultaneous determination of epinephrine and dopamine

A novel taurine modified glassy carbon electrode was prepared by electropolymerization method. The electrochemical behaviors of epinephrine (EP) and dopamine (DA) at the modified electrode were studied by cyclic voltammetry. The modified electrode exhibited enhanced sensitivity and excellent electrochemical discrimination to DA and EP. The cathodic peaks of the two species were well-separated with a potential difference of about 390 mV, so the poly(taurine) modified electrode was used for simultaneous voltammetric measurement of EP and DA by differential pulse voltammetry. Under the optimum conditions, the cathodic peak currents were linear to concentrations of EP and DA in the range of 2.0 × 10⁻⁶ to 6.0 × 10⁻⁴ mol L⁻¹ and 1.0 × 10⁻⁶ to 8.0 × 10⁻⁴ mol L⁻¹, respectively. The detection limits for EP and DA were 3.0 × 10⁻⁷ and 1.0 × 10⁻⁷ mol L⁻¹, respectively. Because the oxidation of ascorbic acid (AA) is an irreversible reaction at modified electrode, the interference of AA for determining EP and DA was eliminated. The modified electrode has been satisfactorily used for the simultaneous determination of EP and DA in pharmaceutical injections.     

Minimalism approach for determination of Cu, Fe and Zn in serum by simultaneous electrothermal atomic absorption spectrometry

Copper, iron and zinc were determined in serum by simultaneous atomic absorption spectrometry (SIMAAS). The minimalism approach was adopted throughout this analytical method, to reduce time, costs, sample, reagent, energy requirements, and residue production. Samples were 80-fold diluted with 0.01% (w/v) Triton X-100+1% (v/v) HNO₃ directly in the autosampler cups. Three strategies were implemented to match the final diluted analyte concentrations with the SIMAAS linear concentration ranges: a reduced 5 μl aliquot of analytical reference or diluted sample solution was introduced into the preheated graphite tube at 100 °C; a super-estimated pyrolysis temperature was chosen for selective zinc volatilization; and a mini argon flow of 50 ml min⁻¹ was used during the atomization step. The pyrolysis and atomization temperatures for the simultaneous heating program were 700 and 2300 °C, respectively. The characteristic masses for copper (26 pg), iron (16 pg), and zinc (2.7 pg) were estimated from the analytical graphs. The detection limits (n=20, 3σ) were 4.0, 2.2, and 0.4 μg l⁻¹ for copper, iron and zinc, respectively. The reliability of the entire procedure was checked with the analysis of Seronorm™ trace elements in serum (Sero AS). Serum samples of five volunteers were analyzed and the recovery tests for additions of 2.0, 2.0 and 1.0 mg l⁻¹ were 100±4, 99±6, and 95±5% for copper, iron and zinc, respectively.     

Application of Multivariate Calibration Techniques to HPLC Data for Quantitative Analysis of a Binary Mixture of Hydrochlorathiazide and Losartan in Tablets

New multivariate approaches have been applied to high-performance liquid chromatography (HPLC) with multiwavelength photodiode-array (PDA) detection. Multivariate calibration techniques such as partial least squares (PLS), principal component regression (PCR), classical least squares (CLS), and inverse least squares (ILS) was subjected to HPLC data for simultaneous quantitative analysis of synthetic binary mixtures and a commercial tablet formulation containing hydrochlorothiazide (HCT) and losartan potassium (LST). The combined use of HPLC and multivariate calibrations has been denoted HPLC–CLS, HPLC–ILS, HPLC–PCR, and HPLC–PLS. Successful chromatographic separation of the two active compounds and enalapril maleate, used as internal standard (IS), was accomplished by means of a 4.6 mm i.d. × 250 mm, 5 m particle, Waters Symmetry C₁₈ reversed-phase column and a mobile phase consisting of 60:40 acetate buffer (0.2 M, pH 4.8)–acetonitrile (v/v, 60:40). HPLC data based on the ratio of analyte peak areas to IS peak area were obtained by PDA detection at five-wavelengths (250, 255, 260, 265, and 270 nm). The HPLC–CLS, HPLC–ILS, HPLC–PCR, and HPLC–PLS calibration plots for hydrochlorothiazide and losartan potassium were constructed separately by using the peak-area ratios corresponding to the concentrations of each active compound. The HPLC multivariate calibrations obtained were tested for different synthetic mixtures containing HCT and LST in the presence of the IS. These multivariate chromatographic methods were also applied to a commercial pharmaceutical dosage form containing HCT and LST. The results obtained from the multivariate calibrations were compared with those obtained by use of another, classical HPLC method using single-wavelength detection.Revised: 29 September 2004 and 4 January 2005     

Simultaneous voltammetric determination of paracetamol and caffeine in pharmaceutical formulations using a boron-doped diamond electrode

A simple and highly selective electrochemical method was developed for the single or simultaneous determination of paracetamol (N-acetyl-p-aminophenol, acetaminophen) and caffeine (3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione) in aqueous media (acetate buffer, pH 4.5) on a boron-doped diamond (BDD) electrode using square wave voltammetry (SWV) or differential pulse voltammetry (DPV). Using DPV with the cathodically pre-treated BDD electrode, a separation of about 550 mV between the peak oxidation potentials of paracetamol and caffeine present in binary mixtures was obtained. The calibration curves for the simultaneous determination of paracetamol and caffeine showed an excellent linear response, ranging from 5.0 × 10⁻⁷ mol L⁻¹ to 8.3 × 10⁻⁵ mol L⁻¹ for both compounds. The detection limits for the simultaneous determination of paracetamol and caffeine were 4.9 × 10⁻⁷ mol L⁻¹ and 3.5 × 10⁻⁸ mol L⁻¹, respectively. The proposed method was successfully applied in the simultaneous determination of paracetamol and caffeine in several pharmaceutical formulations (tablets), with results similar to those obtained using a high-performance liquid chromatography method (at 95% confidence level).     

A simple electroanalytical methodology for the simultaneous determination of dopamine,serotonin and ascorbic acid using an unmodified edge plane pyrolytic graphite electrode

A simple method using an unmodified edge plane pyrolytic graphite electrode (EPPGE) is reported for the simultaneous determination of dopamine (DA), serotonin (ST) and ascorbic acid (AA). The performance of this electrode is superior to other unmodified carbon-based electrodes and also to many modified electrodes in terms of detection limit, sensitivity and peak separation for determination of DA, ST and AA. Using this method, detection limits of 90 nM, 60 nM and 200 nM were obtained for DA, ST and AA respectively. No electrode fouling is observed during a set of experiments and good sensitivity is obtained for the simultaneous determination of DA, ST and AA. The peaks for the three species are well resolved from each other and the electrode is successfully utilised for their determination in standard and real samples.      

Development and validation of a stability-indicating HPLC assay method for simultaneous determination of spironolactone and furosemide in tablet formulation

The objective of the current study was to develop and validate a simple, precise and accurate isocratic stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) assay method for the determination of spironolactone and furosemide in solid pharmaceutical dosage forms. Isocratic RP-HPLC separation was achieved on an SGE 150 × 4.6 mm SS Wakosil II 5C8RS 5-μm column using a mobile phase of acetonitrile-ammonium acetate buffer (50:50, v/v) at a flow rate of 1.0 mL/min. The detection was carried out at 254 nm using a photodiode array detector. The drug was subject to oxidation, hydrolysis, photolysis and heat to apply stress conditions. The method was validated for specificity, linearity, precision, accuracy, robustness and solution stability. The method was found to be linear in the drug concentration range of 40-160 μg/mL with correlation coefficients of 0.9977 and 0.9953 for spironolactone and furosemide, respectively. The precision (relative standard deviation; RSD) among a six-sample preparation was 0.87% and 1.1% for spironolactone and furosemide, respectively. Repeatability and intermediate precision (RSD) among a six-sample preparation were 0.46% and 0.20% for spironolactone and furosemide, respectively. The accuracy (recovery) was between 98.05 and 100.17% and 99.07 and 100.58% for spironolactone and furosemide, respectively. Degradation products produced as a result of stress studies did not interfere with the detection of spironolactone and furosemide; therefore, the assay can be considered to be stability-indicating.     

A simple and precise conductometric method for the determination of losartan in pharmaceutical products

Losartan is an antihypertensive agent that lost its patent protection in 2010, and, consequently, it has been available in generic form. The latter motivated the search for a rapid and precise alternative method. Here, a simple conductometric titration in aqueous medium is described for the losartan analysis in pharmaceutical formulations. The first step of the titration occurs with the protonation of losartan producing a white precipitate and resulting in a slow increase in conductivity. When the protonation stage is complete, a sharp increase in conductivity occurs which was determined to be due to the presence of excess of acid. The titrimetric method was applied to the determination of losartan in pharmaceutical products and the results are comparable with values obtained using a chromatographic method recommended by the United States Pharmacopoeia. The relative standard deviation for successive measurements of a 125 mg L^?1 (2.71×10^?4 mol L^?1) losartan solution was approximately 2%. Recovery study in tablet samples ranged between 99 and 102.4%. The procedure is fast, simple, and represents an attractive alternative for losartan quantification in routine analysis. In addition, it avoids organic solvents, minimizes the risk of exposure to the operator, and the waste treatment is easier compared to classical chromatographic methods.